Publications by authors named "Ana Rondon"

26 Publications

  • Page 1 of 1

Histone H3E73Q and H4E53A mutations cause recombinogenic DNA damage.

Microb Cell 2020 Apr 24;7(7):190-198. Epub 2020 Apr 24.

Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Universidad de Sevilla-CSIC-Universidad Pablo de Olavide, Seville, Spain.

The stability and function of eukaryotic genomes is closely linked to histones and to chromatin structure. The state of the chromatin not only affects the probability of DNA to undergo damage but also DNA repair. DNA damage can result in genetic alterations and subsequent development of cancer and other genetic diseases. Here, we identified two mutations in conserved residues of histone H3 and histone H4 (H3E73Q and H4E53A) that increase recombinogenic DNA damage. Our results suggest that the accumulation of DNA damage in these histone mutants is largely independent on transcription and might arise as a consequence of problems occurring during DNA replication. This study uncovers the relevance of H3E73 and H4E53 residues in the protection of genome integrity.
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http://dx.doi.org/10.15698/mic2020.07.723DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7328676PMC
April 2020

The THO Complex as a Paradigm for the Prevention of Cotranscriptional R-Loops.

Cold Spring Harb Symp Quant Biol 2019 3;84:105-114. Epub 2020 Jun 3.

Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla-CSIC-Universidad Pablo de Olavide, 41092 Seville, Spain.

Different proteins associate with the nascent RNA and the RNA polymerase (RNAP) to catalyze the transcription cycle and RNA export. If these processes are not properly controlled, the nascent RNA can thread back and hybridize to the DNA template forming R-loops capable of stalling replication, leading to DNA breaks. Given the transcriptional promiscuity of the genome, which leads to large amounts of RNAs from mRNAs to different types of ncRNAs, these can become a major threat to genome integrity if they form R-loops. Consequently, cells have evolved nuclear factors to prevent this phenomenon that includes THO, a conserved eukaryotic complex acting in transcription elongation and RNA processing and export that upon inactivation causes genome instability linked to R-loop accumulation. We revise and discuss here the biological relevance of THO and a number of RNA helicases, including the THO partner UAP56/DDX39B, as a paradigm of the cellular mechanisms of cotranscriptional R-loop prevention.
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http://dx.doi.org/10.1101/sqb.2019.84.039594DOI Listing
June 2020

R-Loops as Promoters of Antisense Transcription.

Mol Cell 2019 11;76(4):529-530

Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Universidad de Sevilla- CSIC, Seville, Spain. Electronic address:

The study by Tan-Wong et al. (2019) in this issue of Molecular Cell reveals a capacity of R-loops to promote antisense transcription expanding our view of the features that a DNA region may have to act as a promoter.
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http://dx.doi.org/10.1016/j.molcel.2019.11.001DOI Listing
November 2019

Depletion of the MFAP1/SPP381 Splicing Factor Causes R-Loop-Independent Genome Instability.

Cell Rep 2019 08;28(6):1551-1563.e7

Centro Andaluz de Biología Molecular y Medicina Regenerativa-CABIMER, Universidad de Sevilla-CSIC-Universidad Pablo de Olavide, Seville 41092, Spain. Electronic address:

THO/TREX is a conserved complex with a role in messenger ribonucleoprotein biogenesis that links gene expression and genome instability. Here, we show that human THO interacts with MFAP1 (microfibrillar-associated protein 1), a spliceosome-associated factor. Interestingly, MFAP1 depletion impairs cell proliferation and genome integrity, increasing γH2AX foci and DNA breaks. This phenotype is not dependent on either transcription or RNA-DNA hybrids. Mutations in the yeast orthologous gene SPP381 cause similar transcription-independent genome instability, supporting a conserved role. MFAP1 depletion has a wide effect on splicing and gene expression in human cells, determined by transcriptome analyses. MFAP1 depletion affects a number of DNA damage response (DDR) genes, which supports an indirect role of MFAP1 on genome integrity. Our work defines a functional interaction between THO and RNA processing and argues that splicing factors may contribute to genome integrity indirectly by regulating the expression of DDR genes rather than by a direct role.
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http://dx.doi.org/10.1016/j.celrep.2019.07.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6693559PMC
August 2019

Increased Working Memory Load in a Dual-Task Design Impairs Nonverbal Social Encoding in Children with High and Low Attention-Deficit/Hyperactivity Disorder Symptoms.

Child Psychiatry Hum Dev 2020 02;51(1):127-137

Department of Psychology, The University of Alabama, Box 870348, Tuscaloosa, AL, 35487, USA.

Children with attention-deficit/hyperactivity disorder (ADHD) are known to have difficulty with peer relations, though the mechanisms by which these children struggle with interpersonal relationships are not well known. The current study examined the relation between working memory (WM) and the encoding of nonverbal social cues using a dual-task paradigm tested in children with High and Low ADHD symptoms. A total of 40 children were recruited (20 High ADHD; 20 Low ADHD) and completed computerized tasks of social encoding and WM in both single- and dual-task conditions. A series of repeated measures mixed-model ANOVAs revealed that both children with High ADHD and Low ADHD performed significantly worse during the dual-task condition compared to the single task conditions. Also, children with High ADHD had significantly lower performance than Low ADHD children on task-based social encoding and WM. This study supports the role of WM in nonverbal social encoding in children.
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http://dx.doi.org/10.1007/s10578-019-00915-3DOI Listing
February 2020

What causes an RNA-DNA hybrid to compromise genome integrity?

DNA Repair (Amst) 2019 09 8;81:102660. Epub 2019 Jul 8.

Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Universidad de Sevilla-CSIC-Universidad Pablo de Olavide, 41092, Seville, Spain. Electronic address:

Transcription is a source of genome instability that stimulates mutation and recombination. Part of the damage produced by transcription is mediated by R-loops, non-B DNA structures that normally form by the re-annealing of the nascent RNA with the template DNA outside the catalytic center of the RNA polymerase, displacing the non-template strand. Recent discoveries have revealed that R-loops might not be harmful by themselves. Instead, chromatin compaction triggered by these structures seems necessary, as deduced from the histone modifications frequently found associated with harmful R-loops. Remarkably, hybrids may also become harmful if stabilized by specific RNA binding proteins, one example of which is the yeast Yra1. We discuss here the possible mechanisms by which cells may stabilize R-loops and the consequences on transcription-replication conflicts and telomere homeostasis.
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http://dx.doi.org/10.1016/j.dnarep.2019.102660DOI Listing
September 2019

Social Functioning in Youth with Attention-Deficit/Hyperactivity Disorder and Sluggish Cognitive Tempo.

Yale J Biol Med 2019 03 25;92(1):29-35. Epub 2019 Mar 25.

University of Alabama, Department of Psychology, Tuscaloosa, AL.

The current review summarizes the research to date on social functioning for youth with attention-deficit/hyperactivity disorder (ADHD) with a focus on three key domains: peer rejection, friendship, and social information processing. The review extends past reviews by examining the research to date on how the presence of sluggish cognitive tempo (SCT) symptoms, a common correlate of ADHD, affects the social presentation of youth with ADHD. Overall, youth with ADHD show significant difficulty with peer rejection, forming and maintaining friendships, and abnormalities in how they process and respond to social information. Further, the presence of SCT symptoms results in great social withdrawal and isolation. Future studies are needed to better understand the social difficulties of youth with ADHD, particularly using experimental approaches that can manipulate and isolate mechanisms within the social information processing model. In addition, novel intervention approaches are needed to more effectively ameliorate the social difficulties of youth with ADHD and those with co-occurring SCT symptoms.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6430168PMC
March 2019

Yra1-bound RNA-DNA hybrids cause orientation-independent transcription-replication collisions and telomere instability.

Genes Dev 2018 07 28;32(13-14):965-977. Epub 2018 Jun 28.

Andalusian Center of Molecular Biology and Regenerative Medicine (CABIMER), Universidad de Sevilla-Consejo Superior de Investigaciones Científicas (CSIC)-Universidad Pablo de Olavide, 41092 Seville, Spain.

R loops are an important source of genome instability, largely due to their negative impact on replication progression. Yra1/ALY is an abundant RNA-binding factor conserved from yeast to humans and required for mRNA export, but its excess causes lethality and genome instability. Here, we show that, in addition to ssDNA and ssRNA, Yra1 binds RNA-DNA hybrids in vitro and, when artificially overexpressed, can be recruited to chromatin in an RNA-DNA hybrid-dependent manner, stabilizing R loops and converting them into replication obstacles in vivo. Importantly, an excess of Yra1 increases R-loop-mediated genome instability caused by transcription-replication collisions regardless of whether they are codirectional or head-on. It also induces telomere shortening in telomerase-negative cells and accelerates senescence, consistent with a defect in telomere replication. Our results indicate that RNA-DNA hybrids form transiently in cells regardless of replication and, after stabilization by excess Yra1, compromise genome integrity, in agreement with a two-step model of R-loop-mediated genome instability. This work opens new perspectives to understand transcription-associated genome instability in repair-deficient cells, including tumoral cells.
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http://dx.doi.org/10.1101/gad.311274.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6075034PMC
July 2018

Sleep, Internalizing Problems, and Social Withdrawal: Unique Associations in Clinic-Referred Youth With Elevated Sluggish Cognitive Tempo Symptoms.

J Atten Disord 2020 02 7;24(4):524-534. Epub 2018 Feb 7.

Virginia Polytechnic Institute and State University, Blacksburg, USA.

We compared clinic-referred youth with ADHD + sluggish cognitive tempo (SCT; = 34), ADHD Only ( = 108), and SCT Only ( = 22) on demographics, co-occurring symptomatology, comorbid diagnoses, and social functioning. In total, 164 youth (age = 6-17 years, = 9.97) and their parent(s) presented to an outpatient clinic for a psychoeducational assessment. Between-group analyses and regressions were used to examine study variables. SCT groups were older and exhibited more parent-reported internalizing problems, externalizing problems, sleep problems, and social withdrawal on the Child Behavior Checklist. No significant differences emerged between groups on the Teacher Report Form. Regression analyses involving multiple covariates revealed that SCT symptoms were uniquely related to social withdrawal but not general social problems. Based on parent report, SCT symptoms have a unique relationship with internalizing problems, sleep problems, and social withdrawal. Future research should explore correlates of SCT in youth using multiple informants.
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http://dx.doi.org/10.1177/1087054718756197DOI Listing
February 2020

An EEG Study of Children With and Without ADHD Symptoms: Between-Group Differences and Associations With Sluggish Cognitive Tempo Symptoms.

J Atten Disord 2020 05 11;24(7):1002-1010. Epub 2017 Aug 11.

The University of Alabama, Tuscaloosa, USA.

We examined differences between those with and without ADHD symptoms on resting state electroencephalography (EEG) indices and unique relations with sluggish cognitive tempo (SCT) symptoms. Children with ADHD symptoms ( = 21) and healthy controls ( = 20) were assessed using rating scales, a neuropsychological task measuring sustained attention and inhibitory control, and EEG activity during a resting state period. Between-group, correlational, and regression analyses were conducted. Large differences (particularly for theta/beta ratio in frontal and frontocentral regions) were found on EEG measures between those with and without ADHD symptoms. While ADHD and SCT symptoms both related to sustained attention on a computerized task, only ADHD symptoms were related to frontal and frontocentral theta/beta ratio. Results support the conclusion that ADHD symptoms are strongly associated with theta/beta ratio in frontal and frontocentral regions. Future studies should explore unique neurophysiological correlates of SCT.
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http://dx.doi.org/10.1177/1087054717723986DOI Listing
May 2020

Histone Mutants Separate R Loop Formation from Genome Instability Induction.

Mol Cell 2017 Jun;66(5):597-609.e5

Centro Andaluz de Biología Molecular y Medicina Regenerativa-CABIMER, Universidad de Sevilla-CSIC-Universidad Pablo de Olavide, Seville 41092, Spain. Electronic address:

R loops have positive physiological roles, but they can also be deleterious by causing genome instability, and the mechanisms for this are unknown. Here we identified yeast histone H3 and H4 mutations that facilitate R loops but do not cause instability. R loops containing single-stranded DNA (ssDNA), versus RNA-DNA hybrids alone, were demonstrated using ssDNA-specific human AID and bisulfite. Notably, they are similar size regardless of whether or not they induce genome instability. Contrary to mutants causing R loop-mediated instability, these histone mutants do not accumulate H3 serine-10 phosphate (H3S10-P). We propose a two-step mechanism in which, first, an altered chromatin facilitates R loops, and second, chromatin is modified, including H3S10-P, as a requisite for compromising genome integrity. Consistently, these histone mutations suppress the high H3S10 phosphorylation and genomic instability of hpr1 and sen1 mutants. Therefore, contrary to what was previously believed, R loops do not cause genome instability by themselves.
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http://dx.doi.org/10.1016/j.molcel.2017.05.014DOI Listing
June 2017

Immediate effect of laryngeal manual therapy in dysphonic individuals.

Codas 2016 Jan-Feb;28(1):59-65

Departamento de Fonoaudiologia, Faculdade de Odontologia de Bauru, Universidade de São Paulo, Bauru, SP, Brazil.

Purposes: To investigate the immediate effect of Laryngeal Manual Therapy (LMT) in musculoskeletal pain, in voice and sensations referred to individuals with behavioral dysphonia and individuals without it.

Methods: 30 individuals ranging from 18 to 45 years old were selected and sorted into two groups: the dysphonic group (DG) - 15 individuals with functional or organofunctional dysphonia, and the control group (CG) - 15 individuals without vocal complaints and with non-impaired voices. The individuals answered a pain questionnaire and their voices were subsequently registered. The initial evaluation was repeated after the LMT. The LMT was applied for 20 minutes. After the LMT, the individuals were self-evaluated in terms of sensations in their voices, larynxes, articulations and respiration.

Results: After the application of LMT, the DG reported significant improvement of pain in the following areas: temporal, larynx, posterior neck, wrists/hands/fingers, upper and lower back, hip/thigh, which did not occur in CG. The perceptual analysis of the vowel /a/ revealed no significant difference in any parameter in both groups after the LMT. The analysis of the speech showed that there was an increase of the roughness parameter after the application of LMT just in the DG. The DG individuals reported better sensations in the larynx and articulations after the submission to LMT, which did not occur in CG.

Conclusion: this study clarified that TML immediately reduces the intensity of corporal pain in dysphonic individuals, which did not occur in individuals without any vocal impairments. Although the perceptual analysis reveals an increase of the roughness in the quality of the voice, positive sensation in the larynx and articulation were reported by dysphonic individuals after the application of TML.
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http://dx.doi.org/10.1590/2317-1782/20162015089DOI Listing
November 2016

ADHD Dimensions and Sluggish Cognitive Tempo Symptoms in Relation to Self-Report and Laboratory Measures of Neuropsychological Functioning in College Students.

J Atten Disord 2017 Jun 16;21(8):673-683. Epub 2014 Dec 16.

2 Cincinnati Children's Hospital Medical Center, OH, USA.

Objective: This study examined ADHD and sluggish cognitive tempo (SCT) symptoms in relation to self-report and laboratory measures of neuropsychological functioning in college students.

Method: College students ( N = 298, aged 17-25, 72% female) completed self-reports of ADHD, SCT, depression, sleep, functional impairment, and executive functioning (EF). Participants also completed a visual working memory task, a Stroop test, and the Conners' Continuous Performance Test-II (CPT-II).

Results: ADHD inattentive and SCT symptoms were strong predictors of self-reported EF, with inattention the strongest predictor of Time Management and Motivation and SCT the strongest predictor of Self-Organization/Problem Solving. SCT (but not inattention) was associated with Emotion Regulation. No relationships were found between self-reported symptoms and laboratory task performance. Between-group analyses were largely consistent with regression analyses.

Conclusion: Self-reported ADHD and SCT symptoms are strongly associated with college students' self-reported EF, but relationships with laboratory task measures of neuropsychological functioning are limited.
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http://dx.doi.org/10.1177/1087054714560821DOI Listing
June 2017

R loops are linked to histone H3 S10 phosphorylation and chromatin condensation.

Mol Cell 2013 Nov 7;52(4):583-90. Epub 2013 Nov 7.

Department of Molecular Biology, Universidad de Sevilla-CSIC, Avenida Américo Vespucio, 41092 Seville, Spain.

R loops are transcription byproducts that constitute a threat to genome integrity. Here we show that R loops are tightly linked to histone H3 S10 phosphorylation (H3S10P), a mark of chromatin condensation. Chromatin immunoprecipitation (ChIP)-on-chip (ChIP-chip) analyses reveal H3S10P accumulation at centromeres, pericentromeric chromatin, and a large number of active open reading frames (ORFs) in R-loop-accumulating yeast cells, better observed in G1. Histone H3S10 plays a key role in maintaining genome stability, as scored by ectopic recombination and plasmid loss, Rad52 foci, and Rad53 checkpoint activation. H3S10P coincides with the presence of DNA-RNA hybrids, is suppressed by ribonuclease H overexpression, and causes reduced accessibility of restriction endonucleases, implying a tight connection between R loops, H3S10P, and chromatin compaction. Such histone modifications were also observed in R-loop-accumulating Caenorhabditis elegans and HeLa cells. We therefore provide a role of RNA in chromatin structure essential to understand how R loops modulate genome dynamics.
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http://dx.doi.org/10.1016/j.molcel.2013.10.006DOI Listing
November 2013

New clues to understand the role of THO and other functionally related factors in mRNP biogenesis.

Biochim Biophys Acta 2012 Jun 20;1819(6):514-20. Epub 2011 Dec 20.

Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla-CSIC, Av Américo Vespucio s/n, 41092 Sevilla, Spain. rlvarp@is/es

Coupling of transcription with mRNA processing and export has been shown to be relevant to efficient gene expression. A number of studies have determined that THO/TREX, a nuclear protein complex conserved from yeast to humans, plays an important role in mRNP biogenesis connecting transcription elongation, mRNA export and preventing genetic instability. Recent data indicates that THO could be relevant to different mRNA processing steps, including the 3'-end formation, transcript release and export. Novel connections of THO to proteins related to the splicing machinery, provide new views about possible functions of THO in mRNP biogenesis. In this review, we summarize the previous and new results concerning the impact of THO in transcription and its biological implications, with a special emphasis on the relationship with THSC/TREX-2 and other functionally related factors involved in mRNA biogenesis and export. The emerging picture presents THO as a dynamic complex interacting with the nascent RNA and with different factors connecting nuclear functions necessary for mRNP biogenesis with genome integrity, cellular homeostasis and development. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing.
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http://dx.doi.org/10.1016/j.bbagrm.2011.11.012DOI Listing
June 2012

A novel assay identifies transcript elongation roles for the Nup84 complex and RNA processing factors.

EMBO J 2011 May 8;30(10):1953-64. Epub 2011 Apr 8.

Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla-CSIC, Sevilla, Spain.

To clarify the role of a number of mRNA processing factors in transcription elongation, we developed an in vivo assay for direct analysis of elongation on chromatin. The assay relies on two substrates containing two G-less cassettes separated by either a long and GC-rich or a short and GC-poor DNA sequence (G-less-based run-on (GLRO) assay). We demonstrate that PAF, THSC/TREX-2, SAGA, the exosome component Rrp6 and two subunits of cleavage factor IA (Rna14 and Rna15) are required for efficient transcription elongation, in contrast to some results obtained using other assays. Next, we undertook a mutant screen and found out that the Nup84 nucleoporin complex is also required for transcription elongation, as confirmed by the GLRO assay and RNA polymerase II chromatin immunoprecipitations. Therefore, in addition to showing that the GLRO assay is a sensitive and reliable method for the analysis of elongation in vivo, this study provides evidence for a new role of the Nup84 complex and a number of mRNA processing factors in transcription elongation that supports a connection of pre-mRNA processing and nuclear export with transcription elongation.
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http://dx.doi.org/10.1038/emboj.2011.109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3098481PMC
May 2011

Yeast Sen1 helicase protects the genome from transcription-associated instability.

Mol Cell 2011 Jan;41(1):21-32

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.

Sen1 of S. cerevisiae is a known component of the NRD complex implicated in transcription termination of nonpolyadenylated as well as some polyadenylated RNA polymerase II transcripts. We now show that Sen1 helicase possesses a wider function by restricting the occurrence of RNA:DNA hybrids that may naturally form during transcription, when nascent RNA hybridizes to DNA prior to its packaging into RNA protein complexes. These hybrids displace the nontranscribed strand and create R loop structures. Loss of Sen1 results in transient R loop accumulation and so elicits transcription-associated recombination. SEN1 genetically interacts with DNA repair genes, suggesting that R loop resolution requires proteins involved in homologous recombination. Based on these findings, we propose that R loop formation is a frequent event during transcription and a key function of Sen1 is to prevent their accumulation and associated genome instability.
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http://dx.doi.org/10.1016/j.molcel.2010.12.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3314950PMC
January 2011

The interface between transcription and mRNP export: from THO to THSC/TREX-2.

Biochim Biophys Acta 2010 Aug 19;1799(8):533-8. Epub 2010 Jun 19.

Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Universidad de Sevilla-CSIC, Av. Americo Vespucio s/n, 41092 Sevilla, Spain.

Eukaryotic gene expression is a multilayer process covering transcription to post-translational protein modifications. As the nascent pre-mRNA emerges from the RNA polymerase II (RNAPII), it is packed in a messenger ribonucleoparticle (mRNP) whose optimal configuration is critical for the normal pre-mRNA processing and mRNA export, mRNA integrity as well as for transcription elongation efficiency. The interplay between transcription and mRNP formation feeds forward and backward and involves a number of conserved factors, from THO to THSC/TREX-2, which in addition have a unique impact on transcription-dependent genome instability. Here we review our actual knowledge of the role that these factors play at the interface between transcription and mRNA export in the model organism Saccharomyces cerevisiae.
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http://dx.doi.org/10.1016/j.bbagrm.2010.06.002DOI Listing
August 2010

Fail-safe transcriptional termination for protein-coding genes in S. cerevisiae.

Mol Cell 2009 Oct;36(1):88-98

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.

Transcription termination of RNA polymerase II (Pol II) on protein-coding genes in S. cerevisiae relies on pA site recognition by 3' end processing factors. Here we demonstrate the existence of two alternative termination mechanisms that rescue polymerases failing to disengage from the template at pA sites. One of these fail-safe mechanisms is mediated by the NRD complex, similar to termination of short noncoding genes. The other termination mechanism is mediated by Rnt1 cleavage of the nascent transcript. Both fail-safe termination mechanisms trigger degradation of readthrough transcripts by the exosome. However, Rnt1-mediated termination can also enhance the usage of weak pA signals and thereby generate functional mRNA. We propose that these alternative Pol II termination pathways serve the dual function of avoiding transcription interference and promoting rapid removal of aberrant transcripts.
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http://dx.doi.org/10.1016/j.molcel.2009.07.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2779338PMC
October 2009

Nuclear roadblocks for mRNA export.

Cell 2008 Oct;135(2):207-8

Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK.

The THO complex and Sub2 RNA helicase have been shown to function in both transcription and mRNA processing. Rougemaille et al. (2008) now uncover evidence that THO/Sub2 coordinates mRNA processing and nuclear export.
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http://dx.doi.org/10.1016/j.cell.2008.09.053DOI Listing
October 2008

Budding yeast RNA polymerases I and II employ parallel mechanisms of transcriptional termination.

Genes Dev 2008 Apr;22(8):1082-92

Sir William Dunn School of Pathology, Oxford OX1 3RE, United Kingdom.

Both RNA polymerase I and II (Pol I and Pol II) in budding yeast employ a functionally homologous "torpedo-like" mechanism to promote transcriptional termination. For two well-defined Pol II-transcribed genes, CYC1 and PMA1, we demonstrate that both Rat1p exonuclease and Sen1p helicase are required for efficient termination by promoting degradation of the nascent transcript associated with Pol II, following mRNA 3' end processing. Similarly, Pol I termination relies on prior Rnt1p cleavage at the 3' end of the pre-rRNA 35S transcript. This is followed by the combined actions of Rat1p and Sen1p to degrade the Pol I-associated nascent transcript that consequently promote termination in the downstream rDNA spacer sequence. Our data suggest that the previously defined in vitro Pol I termination mechanism involving the action of the Reb1p DNA-binding factor to "road-block" Pol I transcription close to the termination region may have overlooked more complex in vivo molecular processes.
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http://dx.doi.org/10.1101/gad.463408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2335328PMC
April 2008

Molecular evidence indicating that the yeast PAF complex is required for transcription elongation.

EMBO Rep 2004 Jan;5(1):47-53

Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Avd. Reina Mercedes 6, 41012 Sevilla, Spain.

PAF is a five-subunit protein complex composed of Paf1, Cdc73, Leo1, Rtf1 and Ctr9, which was purified from yeast in association with RNA polymerase II and which is believed to function in transcription elongation. However, no direct proof exists for this yet. To assay whether PAF is required in elongation, we determined the in vitro transcription-elongation efficiencies of mutant cell extracts using a DNA template containing two G-less cassettes. paf1Delta or cdc73Delta cell extracts showed reduced transcription-elongation efficiencies (16-18% of the wild-type levels), whereas leo1Delta and rtf1Delta showed wild-type levels. In vivo transcription efficiency was diminished in the four mutants analysed, as determined by their abilities to transcribe lacZ. Our work provides molecular evidence that PAF has a positive role in transcription elongation and is composed of at least two functionally different types of subunits (Paf1-Cdc73 and Leo1-Rtf1).
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http://dx.doi.org/10.1038/sj.embor.7400045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1298956PMC
January 2004

Molecular evidence that the eukaryotic THO/TREX complex is required for efficient transcription elongation.

J Biol Chem 2003 Oct 18;278(40):39037-43. Epub 2003 Jul 18.

Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Avenida Reina Mercedes 6, 41012 Sevilla, Spain.

THO/TREX is a conserved eukaryotic complex formed by the core THO complex plus proteins involved in mRNA metabolism and export such as Sub2 and Yra1. Mutations in any of the THO/TREX structural genes cause pleiotropic phenotypes such as transcription impairment, increased transcription-associated recombination, and mRNA export defects. To assay the relevance of THO/TREX complex in transcription, we performed in vitro transcription elongation assays in mutant cell extracts using supercoiled DNA templates containing two G-less cassettes. With these assays, we demonstrate that hpr1delta, tho2delta, and mft1delta mutants of the THO complex and sub2 mutants show significant reductions in the efficiency of transcription elongation. The mRNA expression defect of hpr1delta mutants was not due to an increase in mRNA decay, as determined by mRNA half-life measurements and mRNA time course accumulation experiments in the absence of Rrp6p exoribonuclease. This work demonstrates that THO and Sub2 are required for efficient transcription elongation, providing further evidence for the coupling between transcription and mRNA metabolism and export.
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http://dx.doi.org/10.1074/jbc.M305718200DOI Listing
October 2003

Molecular evidence for a positive role of Spt4 in transcription elongation.

EMBO J 2003 Feb;22(3):612-20

Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Avd. Reina Mercedes 6, E-41012 Sevilla, Spain.

We have previously shown that yeast mutants of the THO complex have a defect in gene expression, observed as an impairment of lacZ transcription. Here we analyze the ability of mutants of different transcription elongation factors to transcribe lacZ. We found that spt4Delta, like THO mutants, impaired transcription of lacZ and of long and GC-rich DNA sequences fused to the GAL1 promoter. Using a newly developed in vitro transcription elongation assay, we show that Spt4 is required in elongation. There is a functional interaction between Spt4 and THO, detected by the lethality or strong gene expression defect and hyper-recombination phenotypes of double mutants in the W303 genetic background. Our results indicate that Spt4-Spt5 has a positive role in transcription elongation and suggest that Spt4-Spt5 and THO act at different steps during mRNA biogenesis.
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http://dx.doi.org/10.1093/emboj/cdg047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC140732PMC
February 2003

TREX is a conserved complex coupling transcription with messenger RNA export.

Nature 2002 May 28;417(6886):304-8. Epub 2002 Apr 28.

BZH, University of Heidelberg, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany.

The essential yeast proteins Yra1 and Sub2 are messenger RNA export factors that have conserved counterparts in metazoans, designated Aly and UAP56, respectively. These factors couple the machineries that function in splicing and export of mRNA. Here we show that both Yra1 and Sub2 are stoichiometrically associated with the heterotetrameric THO complex, which functions in transcription in yeast. We also show that Sub2 and Yra1 interact genetically with all four components of the THO complex (Tho2, Hpr1, Mft1 and Thp2). Moreover, these components operate in the export of bulk poly(A)(+) RNA as well as of mRNA derived from intronless genes. Both Aly and UAP56 associate with human counterparts of the THO complex. Together, these data define a conserved complex, designated the TREX ('transcription/export') complex. The TREX complex is specifically recruited to activated genes during transcription and travels the entire length of the gene with RNA polymerase II. Our data indicate that the TREX complex has a conserved role in coupling transcription to mRNA export.
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http://dx.doi.org/10.1038/nature746DOI Listing
May 2002